Rubella virus consists of a single stranded polyadenylated genomic RNA of positive polarity. Sequences at the 5' and 3' end of the genomic RNA can form potentially stable stem-loop (SL) structures and have been implicated in viral replication. Previously, we have demonstrated that cellular proteins specifically interact with both the SL structures and the interaction is necessary for translation. In addition, the binding of a 56 kDa cellular protein to the 3'(+) SL RNA of Rubella virus coincides with the onset of negative-strand RNA synthesis. In this study, we have purified this host protein to homogeneity. The purified protein specifically interacts with the 3'(+) SL RNA with similar affinity as that present in the total cell lysate. Tryptic peptide sequence of the protein was determined and the sequences obtained were compared with the gene bank sequences. The amino acid comparison demonstrated that the 60 kDa purified host protein is calreticulin and is 100% homologous to human calreticulin. Further confirmation of the protein identity was obtained by western blot analysis of 3'(+) SL RNA binding proteins by one or two dimensional PAGE using human calreticulin antibody. The domains of calreticulin responsible for its specific interaction with the 3'(+) SL RNA were identified using tryptic peptides of the protein as competitors in RNA gel shift assay. Currently, studies are underway to define the role of calreticulin interaction in Rubella virus RNA replication.